The blood-brain barrier (BBB) is one of the most complicated organs in the body and one that is difficult to adequately model in vitro. Because the BBB plays a vital role in protecting the brain from peripheral agents, it is important to understand how it functions, and how to identify when it is compromised. On the other hand, the development of CNS targeting imaging agents and drugs for neurological disorders is hindered by the great difficulty in breaching the blood-brain barrier, and it would be a major milestone to selectively manipulate the BBB to allow access of beneficial drugs, or allow egress of accumulated toxic species in the brain. Therefore, a comprehensive model of the BBB is needed, and this will depend on maintaining all of the cellular and molecular components of the BBB, which can only be accomplished in vivo. Using multiphoton microscopy and brain imaging techniques based on fluorescence, we propose to image all of the known components of the BBB to enable a better understanding of this complex organ. We will apply our tools to models of Alzheimer's disease, where a long standing controversy surrounds the role and integrity of the BBB in the disease. Finally, we will manipulate the BBB to allow imaging of candidate molecular imaging probes to quantitatively assess the localization and concentration of soluble toxic oligomers of Ab in the transgenic mouse models of Alzheimer's disease. This will resolve an important controversy concerning the role of soluble vs insoluble amyloid aggregates in the progression of the disease. Ultimately, these studies will provide insight into the overall functio of the BBB in health and disease. The overall impact of the work will be significant, as the understanding and ability to manipulate the BBB selectively will appeal to neuroscientists from a broad range of disciplines. PUBLIC HEALTH RELEVANCE: This proposal aims to develop the tools to study the intact blood-brain barrier in the brain of a living mouse. Models of the blood-brain barrier in vitro cannot recapitulate the complexity of this organ, but by developing fluorescent imaging approaches in combination with intravital multiphoton microscopy we will image and interrogate this structure in its entirety. We will then address controversial hypotheses concerning the integrity of the blood-brain barrier in Alzheimer's disease and develop tools to allow molecular imaging probes to quantitatively measure diffusible aggregates of amyloid in the Alzheimer mouse brain for the first time. The results of the work will enable CNS researchers with the tools and knowledge to overcome the blood-brain barrier to allow imaging agents and drugs to reach their targets in the brain.